In eutherian mammals, the placenta is the first functional organ. Our previous work highlights the unusual plasticity of the human organ's specialized epithelial cells[unreadable]cytotrophoblasts (CTBs). For example, they mimic many aspects of endothelial cells (ECs) including their adhesion molecule repertoire and production of VEGF and Ang family members. Recently, we considered the possibility that CTB differentiation might involve mechanisms that regulate not only blood vessel (BV) formation, but more precisely arterial specification. The cell-cell interactions that occur as CTBs invade the uterine wall support this theory. CTBs breach the uterine BVs, thereby diverting maternal blood flow to the placenta. Subsequently, the cells form superficial connections with uterine veins. In contrast, they replace the EC lining of uterine spiral arterioles and intercalate within their muscular walls, a process that involves nearly the entire intrauterine course of these vessels. Thus, CTBs have the molecular machinery to preferentially colonize arteries. At a molecular level CTB progenitors within the placenta express EphB4, which is involved in venous specification. As the cells differentiate/invade they abruptly downregulate expression of this receptor and upregulate ephrinB2 required for arterial development. Accordingly, we examined CTB expression of Notch family members that play upstream regulatory roles in vascular development and arterial EC differentiation. We found that CTBs express a broad repertoire of these molecules and that inhibitors of Notch signaling impaired their invasion and ephrinB2 expression. Thus, we now propose experiments to test the hypothesis that Notch signaling plays a critical role in controlling CTB differentiation/invasion and the cells' ability to form a functional arterial endothelium. Accordingly, the goal of Aim 1 is to perform a detailed analysis of CTB expression of Notch family members. In Aim 2, we will study the functions of molecules that are expressed in a manner that suggests important roles. Thus, at the conclusion of the proposed experiments we will know a great deal more about the unusual process of CTB transformation into EC-like cells, which we think will yield important new insights into mechanisms that are crucial to normal placentation and may be dysregulated in cases of infertility or poor pregnancy outcome. By collaborating with other investigators in this U-54 program, we expect to gain additional insights into these normal and disease processes in terms of trophoblast function.